Summary Mutational data from human cancer imply that the p53 tumor suppressor gene is crucial for limiting tumorigenesis. p53 is a sequences specific DNA binding protein that is induced by DNA damage or oncogenic stress leading to induction of genes that trigger a series of anti-proliferative responses whose contribution to tumor suppression remains controversial. Additionally, p53 can directly or indirectly repress gene expression, though the impact of p53 repressed genes on tumor progression and maintenance is poorly understood. Adding to this complexity, p53 mutations typically involve a point mutation in one allele and a large deletion targeting the other, with emerging data indicating that both of these events promote cancer beyond p53 loss. Our project previously established that apoptosis and cellular senescence an be major modes of p53 action in tumor suppression, and most recently identified a key role for p53 in limiting aberrant self renewal and restricting cellular plasticity during tumorigenesis. Using key technologies developed in our group, we showed that reactivation of endogenous p53 in advanced tumors produces potent anti-tumor effects, and explored mechanisms whereby p53 lesions promote tumorigenesis independent of their effects on wild-type p53, for example, identifying therapeutically actionable effectors of p53 mutant action in pancreas cancer and additional haploinsufficient tumor suppressors encompassed within 17p deletions that cooperate with p53 suppress tumorigenesis. In the current proposal, Project 4 will continue to use innovative genetic and animal modeling technologies to address significant unanswered questions in the p53 field. For example, it embraces and studies the notion that p53 action depends on context, and combines powerful uses novel mosaic mouse models to interrogate mechanisms of p53 mediated tumor suppression in different tissue and genetic settings. Using potent and inducible shRNA technology optimized in the group, it tests the novel hypothesis that genes normally repressed by p53 and aberrantly increased in mutant tumors contribute to tumor maintenance and may include targets that are synthetically lethal to mutant p53. In doing so, it implements unique inducible shRNA transgenic technology to explore the impact of target inhibition in tumor and normal tissues. Finally, the project will develop a streamlined method for producing p53 mutant alleles in mice, and use this to explore the biology of highly frequent but understudied p53 truncating alleles to determine whether they produce have gain of function properties. Each of our proposed Aims is supported by substantial preliminary data and will benefit from interactions with all other projects and cores. Successful completion of the proposed work will substantially understand how p53 suppresses tumorigenesis in vivo, and may point to therapeutic opportunities relevant to a large fraction of human cancers.